Inverse effects of dansylation of red blood cell membrane on band 3 protein-mediated transport of sulphate and chloride.

1. Dansylation of the red cell membrane produces inverse effects on SO(4) (2-) and Cl(-) equilibrium exchange. The former is enhanced by several orders of magnitude (Legrum, Fasold & Passow, 1980), the latter is inhibited. Both effects are potentiated after dansylation in the presence of 2-(4-amino-3-sulphophenyl)-6-methyl-7-benzothiazol sulphonic acid (APMB), a disulphonic acid that combines non-covalently with the 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulphonic acid (H(2)DIDS) binding site of the anion transport protein.2. After dansylation the maximum of the pH dependence of SO(4) (2-) exchange near pH 6.3 is replaced by a plateau. When dansylation is performed in the presence of APMB, the plateau is reached at a much higher level at around pH 7.0 and resembles that observed by Funder & Wieth (1976) for Cl(-).3. The mutual interactions between the transfer site, the H(2)DIDS binding site, and the as yet unidentified danysl chloride binding sites were studied in detail. Occupation of the H(2)DIDS binding site by the non-covalently binding agents 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS), 4,4'-bis(acetamido) stilbene-2,2'-disulphonate (DAS) or APMB inhibit the enhanced SO(4) (2-) exchange across the previously dansylated membrane. The apparent K(I) value remains the same as in untreated membranes for DNDS, is reduced to 1/3 for DAS, and to 1/60 for APMB. Conversely, when dansylation is carried out while the H(2)DIDS binding site is occupied by DNDS, APMB or DAS, the enhancement of SO(4) (2-) exchange (as measured after removal of excess dansyl chloride and the additional agent) is prevented by DNDS, augmented by APMB and not affected by DAS. This suggests that the agents stabilize different conformations of the H(2)DIDS binding site that are associated with different accessibilities of the dansyl chloride binding sites.4. The SO(4) (2-) equilibrium exchange as measured at a fixed Cl(-) concentration is enhanced when the Cl(-) concentration at which dansylation is carried out is increased, indicating allosteric interactions between anion binding and the exposure of the dansyl chloride binding sites.5. The enhanced K(+) efflux from dansylated red cells is independent of the described modifications of the dansylation reaction by APMB, DAS or DNDS, demonstrating that there exists no simple correlation between the changes of anion and cation movements that are induced by dansylation.